Mdm2 and MdmX (also known as Mdm4) are key regulators of the p53 tumor suppressor. Previous work by our lab and by many other groups has determined that these MDM proteins complex with p53 and inhibit p53 functions, with Mdm2 inducing p53 ubiquitination and proteosomal degradation, and MdmX inhibiting p53 transactivation of p53 target gene expression. As such, Mdm2 and MdmX act as critical negative regulators of p53 tumor suppressing functions, and amplification of either the MDM2 or MDMX gene is observed in a significant percentage of human tumors. In this proposal, we summarize the progress we have made in our previous studies on p53 regulation by MDM and other cellular proteins. We also describe our very recent studies examining in vitro the effects of Mdm2 phosphorylation in the regulation of p53 stability and activity, as well as our discovery of p53-independent roles for MdmX in suppressing proliferation and tumorigenesis in p53-null cells and mice. Based upon our preliminary data, we now propose to utilize genetically modified mice bearing amino acid substitutions in Mdm2 in order to examine in vivo the effects of Mdm2 phosphorylation on Mdm2-MdmX-p53 signaling and on p53 functions in development and tumor suppression. We also propose to further characterize p53-independent functions of MdmX in the regulation of cell transformation and chromosomal stability. This research should greatly increase our understanding of the roles of these MDM proteins in regulating cell growth and death, and the functional relationship between these homologues in DNA damage signaling and in p53-dependent and p53-independent suppression of tumorigenesis.